Due to increasing attention to the negative health effects of obesity, a market demand for food and beverage products having alternative nutritional characteristics, including, for example, reduced calorie content, has increased as well. There is a market demand to replace the high-calorie sweeteners typically used in food and beverage products, such as sucrose and high fructose corn syrup (HFCS), with non-nutritive sweeteners. A number of such non-nutritive sweeteners have been identified. Some of these are small proteins which are naturally found in plants. Such proteins are often referred to as sweet proteins.
Brazzein is a sweet protein which can be extracted from the fruit of the West African climbing plant Pentadiplandra brazzeana Baillon (WO9531547). It has been characterized as a monomer protein having a 3-dimensional structure with four evenly spaced disulfide bonds. Three forms of the protein seem to exist in nature differing only at the N-terminal amino acid residue. One corresponds to the predicted 54-amino acid translation product containing a glutamine at its N-terminus. This form has been shown to be short lived as the N-terminal glutamine undergoes natural conversion to pyroglutamate, resulting in the second form. The loss of the N-terminal glutamine or pyroglutamate yields the 53-amino acid form which has been reported to be twice as sweet as the form having an N-terminal pyroglutamate (Lamphear, Barry J. et al. (2005) Plant Biotechnology Journal 3(1): 103-114).
On weight basis, brazzein is 500 to 2,000 times sweeter than sugar. Its sweet perception is quite similar to that of sucrose with a clean sweet taste with lingering aftertaste. Further, it has been shown to be stable over a broad pH range (2.5 to 8) and it can withstand heat which makes it suitable for many industrial food manufacturing processes. As a protein it is safe for diabetics and very soluble in water (>50 mg/ml). Brazzein thus represents a very good alternative to other available low calorie sweeteners.
Natural sourcing from P. brazzeana is difficult and expensive, though, and therefore alternatives for sourcing of the protein are being searched for. Brazzein can be chemically synthesised, which is useful for production of, e.g., variants in small scale, but not suitable for large scale production. Heterologous production in E. coli of brazzein or variants of brazzein has been described (see, e.g., Assadi-Porter, F. M. et al. (2000) Arch. Biochem. Biophys. 376(2): 252-258; WO2008112475). The bacterial system is ideal for structural investigations because it is easy to manipulate genetically and well suited for isotopic labelling. Expression of plant proteins from E. coli may be performed using the advantage of forming inclusion bodies inside the cell that can quite easily be separated from host proteins. However, proteins unfold and precipitate in inclusion bodies, and controlling correct S—S bond formation after resolubilization and refolding is not always easy (see, e.g., Tamás and Shewry (2006) Journal of Cereal Science 43: 259-274). Also, heterologous protein expression using inclusion bodies normally requires a number of purification steps which make such systems less suitable for industrial scale production.
Genetic engineering into plants, such as maize or wheat, has also been described. Brazzein-containing germ flour from maize has been demonstrated useful as a low-intensity sweetener providing a low-calorie alternative to sucrose, which also gives the intrinsic bulking properties necessary to replace the volume lost on removal of sugar. Also, a high-intensity sweetener based on corn-expressed brazzein could potentially be provided from enrichment of such material, which would extend the range of product applications (Lamphear, Barry J. et al. (2005) Plant Biotechnology Journal 3(1): 103-114).
Commercial-scale production of purified brazzein to be used as high-intensity sweetener may be more economic from a microbial system, though. But, in general, industrial production of plant proteins from microbial systems is not straightforward, and obtaining a correctly folded plant protein in high yield from a microorganism may be challenging and is not always possible. One factor which may complicate heterologous expression of brazzein from a microbial system is the small size of the protein. Another factor is the existence of numerous cysteine residues because of, e.g., the possibility of non-native S—S bond formation, possibly leading to loss of function or to intermolecular aggregation, e.g., during secretion. Thaumatin, which is another sweet protein originating from a plant, has been successfully expressed intracellularly in yeast in considerable quantities; however the yeast cells lacked the ability to process this molecule into a functional protein having sweet taste (Lee, J.-H. et al. (1988) Biochemistry 27: 5101-5107).
One purpose for the present inventors has been to identify a method for production of brazzein to be used as a high-intensity sweetener which can be performed in industrial scale. Industrial scale production at low cost requires that the protein is expressed in high yield and can easily be purified in a functionally folded 3-dimensional form possessing the same sweet phenotype as the natural product.